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Annie Andrieux - One of the best experts on this subject based on the ideXlab platform.
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Structural basis of tubulin Detyrosination by the vasohibin-SVBP enzyme complex.
Nature Structural & Molecular Biology, 2019Co-Authors: Na Wang, Leticia Peris, Christophe Bosc, Sung Ryul Choi, Benoit Boulan, Natacha Olieric, Hongyu Bao, Fatma Krichen, Liu Chen, Annie AndrieuxAbstract:Vasohibins are tubulin tyrosine carboxypeptidases that are important in neuron physiology. We examined the crystal structures of human vasohibin 1 and 2 in complex with small vasohibin-binding protein (SVBP) in the absence and presence of different inhibitors and a C-terminal α-tubulin peptide. In combination with functional data, we propose that SVBP acts as an activator of vasohibins. An extended groove and a distinctive surface residue patch of vasohibins define the specific determinants for recognizing and cleaving the C-terminal tyrosine of α-tubulin and for binding microtubules, respectively. The vasohibin-SVBP interaction and the ability of the enzyme complex to associate with microtubules regulate axon specification of neurons. Our results define the structural basis of tubulin Detyrosination by vasohibins and show the relevance of this process for neuronal development. Our findings offer a unique platform for developing drugs against human conditions with abnormal tubulin tyrosination levels, such as cancer, heart defects and possibly brain disorders.
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Cap-Gly proteins at microtubule plus ends: is EB1 Detyrosination involved?
PLOS ONE, 2012Co-Authors: Anouk Bosson, Didier Job, Annie Andrieux, Jean-marc Soleilhac, Odile Valiron, Marie-jo MoutinAbstract:Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids −EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can Detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that Detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin.
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Cap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination
2012Co-Authors: Anouk Bosson, Annie Andrieux, Jean-marc Soleilhac, Odile Valiron, Marie-jo MoutinAbstract:Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with a-tubulin and EB1 through their C-terminal amino acids 2EEY. Detyrosination (cleavage of the terminal tyrosine) of a-tubulin by tubulincarboxypeptidase abolishes CLIP170 binding. Can Detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of a-tubulin was only barely detyrosinated. Our results indicate that Detyrosination regulates CLIPs interaction with a-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin
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Motor-dependent microtubule disassembly driven by tubulin tyrosination
Journal of Cell Biology, 2009Co-Authors: Leticia Peris, Michael Wagenbach, Laurence Lafanechère, Jacques Brocard, Ayana T. Moore, Frank Kozielski, Didier Job, Linda Wordeman, Annie AndrieuxAbstract:In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of alpha-tubulin. The significance of this selective Detyrosination of MTs is not understood. In this study, we report that tubulin Detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)-inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the Detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.
Leticia Peris - One of the best experts on this subject based on the ideXlab platform.
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Defective tubulin Detyrosination causes structural brain abnormalities with cognitive deficiency in humans and mice.
Human Molecular Genetics, 2019Co-Authors: Alistair T. Pagnamenta, Leticia Peris, Christophe Bosc, Pierre Heemeryck, Hilary C. Martin, Ivy Uszynski, Sylvie Gory-fauré, Simon Couly, Charu Deshpande, Ata SiddiquiAbstract:Reversible Detyrosination of tubulin, the building block of microtubules, is crucial for neuronal physiology. Enzymes responsible for Detyrosination were recently identified as complexes of vasohibins (VASHs) one or two with small VASH-binding protein (SVBP). Here we report three consanguineous families, each containing multiple individuals with biallelic inactivation of SVBP caused by truncating variants (p.Q28* and p.K13Nfs*18). Affected individuals show brain abnormalities with microcephaly, intellectual disability and delayed gross motor and speech development. Immunoblot testing in cells with pathogenic SVBP variants demonstrated that the encoded proteins were unstable and non-functional, resulting in a complete loss of VASH Detyrosination activity. Svbp knockout mice exhibit drastic accumulation of tyrosinated tubulin and a reduction of detyrosinated tubulin in brain tissue. Similar alterations in tubulin tyrosination levels were observed in cultured neurons and associated with defects in axonal differentiation and architecture. Morphological analysis of the Svbp knockout mouse brains by anatomical magnetic resonance imaging showed a broad impact of SVBP loss, with a 7% brain volume decrease, numerous structural defects and a 30% reduction of some white matter tracts. Svbp knockout mice display behavioural defects, including mild hyperactivity, lower anxiety and impaired social behaviour. They do not, however, show prominent memory defects. Thus, SVBP-deficient mice recapitulate several features observed in human patients. Altogether, our data demonstrate that deleterious variants in SVBP cause this neurodevelopmental pathology, by leading to a major change in brain tubulin tyrosination and alteration of microtubule dynamics and neuron physiology.
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Structural basis of tubulin Detyrosination by the vasohibin-SVBP enzyme complex.
Nature Structural & Molecular Biology, 2019Co-Authors: Na Wang, Leticia Peris, Christophe Bosc, Sung Ryul Choi, Benoit Boulan, Natacha Olieric, Hongyu Bao, Fatma Krichen, Liu Chen, Annie AndrieuxAbstract:Vasohibins are tubulin tyrosine carboxypeptidases that are important in neuron physiology. We examined the crystal structures of human vasohibin 1 and 2 in complex with small vasohibin-binding protein (SVBP) in the absence and presence of different inhibitors and a C-terminal α-tubulin peptide. In combination with functional data, we propose that SVBP acts as an activator of vasohibins. An extended groove and a distinctive surface residue patch of vasohibins define the specific determinants for recognizing and cleaving the C-terminal tyrosine of α-tubulin and for binding microtubules, respectively. The vasohibin-SVBP interaction and the ability of the enzyme complex to associate with microtubules regulate axon specification of neurons. Our results define the structural basis of tubulin Detyrosination by vasohibins and show the relevance of this process for neuronal development. Our findings offer a unique platform for developing drugs against human conditions with abnormal tubulin tyrosination levels, such as cancer, heart defects and possibly brain disorders.
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vasohibins svbp are tubulin carboxypeptidases tcps that regulate neuron differentiation
Science, 2017Co-Authors: Leticia Peris, Anouk Bosson, Christophe Bosc, Pierre Heemeryck, Chrystelle AillaudAbstract:Reversible Detyrosination of α-tubulin is crucial to microtubule dynamics and functions, and defects have been implicated in cancer, brain disorganization, and cardiomyopathies. The identity of the tubulin tyrosine carboxypeptidase (TCP) responsible for Detyrosination has remained unclear. We used chemical proteomics with a potent irreversible inhibitor to show that the major brain TCP is a complex of vasohibin-1 (VASH1) with the small vasohibin binding protein (SVBP). VASH1 and its homolog VASH2, when complexed with SVBP, exhibited robust and specific Tyr/Phe carboxypeptidase activity on microtubules. Knockdown of vasohibins or SVBP and/or inhibitor addition in cultured neurons reduced detyrosinated α-tubulin levels and caused severe differentiation defects. Furthermore, knockdown of vasohibins disrupted neuronal migration in developing mouse neocortex. Thus, vasohibin/SVBP complexes represent long-sought TCP enzymes.
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Motor-dependent microtubule disassembly driven by tubulin tyrosination
Journal of Cell Biology, 2009Co-Authors: Leticia Peris, Michael Wagenbach, Laurence Lafanechère, Jacques Brocard, Ayana T. Moore, Frank Kozielski, Didier Job, Linda Wordeman, Annie AndrieuxAbstract:In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of alpha-tubulin. The significance of this selective Detyrosination of MTs is not understood. In this study, we report that tubulin Detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)-inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the Detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.
Marin Barisic - One of the best experts on this subject based on the ideXlab platform.
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α-Tubulin Detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity
Journal of Cell Biology, 2020Co-Authors: Luísa T. Ferreira, Bernardo Orr, Girish Rajendraprasad, António J. Pereira, Carolina Lemos, Joana T. Lima, Claudia Guasch Boldú, Jorge G. Ferreira, Marin BarisicAbstract:Incorrect kinetochore-microtubule attachments during mitosis can lead to chromosomal instability, a hallmark of human cancers. Mitotic error correction relies on the kinesin-13 MCAK, a microtubule depolymerase whose activity in vitro is suppressed by α-tubulin Detyrosination-a posttranslational modification enriched on long-lived microtubules. However, whether and how MCAK activity required for mitotic error correction is regulated by α-tubulin Detyrosination remains unknown. Here we found that detyrosinated α-tubulin accumulates on correct, more stable, kinetochore-microtubule attachments. Experimental manipulation of tubulin tyrosine ligase (TTL) or carboxypeptidase (Vasohibins-SVBP) activities to constitutively increase α-tubulin Detyrosination near kinetochores compromised efficient error correction, without affecting overall kinetochore microtubule stability. Rescue experiments indicate that MCAK centromeric activity was required and sufficient to correct the mitotic errors caused by excessive α-tubulin Detyrosination independently of its global impact on microtubule dynamics. Thus, microtubules are not just passive elements during mitotic error correction, and the extent of α-tubulin Detyrosination allows centromeric MCAK to discriminate correct vs. incorrect kinetochore-microtubule attachments, thereby promoting mitotic fidelity.
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Molecular basis of vasohibins-mediated Detyrosination and its impact on spindle function and mitosis.
Cell Research, 2019Co-Authors: Shanhui Liao, Girish Rajendraprasad, Na Wang, Susana Eibes, Jun Gao, Hongda Huang, Marin BarisicAbstract:α-Tubulin Detyrosination, largely catalyzed by vasohibins, is involved in many microtubule (MT)-related cellular events. In this study, we identified a core heterodimeric complex of human small vasohibin-binding protein (SVBP) and vasohibin 1 (VASH1) (hereafter denoted as SVBP-VASH1) that catalyzes the Detyrosination of a peptide derived from C-terminus of α-tubulin. We further solved the crystal structures of the SVBP-VASH1 heterodimer alone and in complex with either an inhibitor or a mutant substrate peptide. Our structural research, complemented by biochemical and mutagenesis experiments, resulted in identification of the key residues for VASH1 binding to SVBP and α-tubulin substrate. Our in vivo experiments reveal that MT Detyrosination in general, as well as the interactions between SVBP, VASH1, and α-tubulin, are critical for spindle function and accurate chromosome segregation during mitosis. Furthermore, we found that the phenotypes caused by the depletion of vasohibins were largely rescued upon co-depletion of kinesin13/MCAK, suggesting the coordination between the MT depolymerase and MT Detyrosination during mitosis. Thus our work not only provides structural insights into the molecular mechanism of α-tubulin Detyrosination catalyzed by SVBP-bound vasohibins, but also uncovers the key role of vasohibins-mediated MT Detyrosination in spindle morphology and chromosome segregation during mitosis.
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MITOSIS Microtubule Detyrosination guides chromosomes during mitosis
2016Co-Authors: Marin Barisic, Ricardo Silva E Sousa, Suvranta K Tripathy, Maria M Magiera, Anatoly V Zaytsev, Ana Luisa Pereira, Carsten Janke, Ekaterina L. Grishchuk, Helder MaiatoAbstract:Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end–directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational Detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E–dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous Detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E–driven chromosome congression is guided by microtubule Detyrosination. C hromosome congression is the process that leads to the formation of ametaphase plat
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microtubule Detyrosination guides chromosomes during mitosis
Science, 2015Co-Authors: Marin Barisic, Ricardo Silva E Sousa, Suvranta K Tripathy, Maria M Magiera, Anatoly V Zaytsev, Ana Luisa PereiraAbstract:Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end–directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational Detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E–dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous Detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E–driven chromosome congression is guided by microtubule Detyrosination.
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Motility of Kinetochore Kinesin CENP-E is Enhanced by Tubulin Detyrosination
Biophysical Journal, 2015Co-Authors: Suvranta K Tripathy, Helder Maiato, Marin Barisic, Maria M Magiera, Anatoly V Zaytsev, Carsten Janke, Ricardo Silva E Sousa, Ekaterina L. GrishchukAbstract:Targeted transport by intracellular motors can be regulated by posttranslational modifications of polymerized tubulins in the microtubule tracks, but little is known about such effects for motors that drive chromosome motions during mitosis. Microtubules that form mitotic spindle are differentially modified at the C-terminal residue of α-tubulin: polymers that point to the spindle equator, but not the astral microtubules, are preferentially detyrosinated. Here we examine the influence of tubulin Detyrosination on CENP-E, the kinetochore-localized kinesin-7 that transports pole-proximal chromosomes to the spindle equator. We polymerized purified human tubulins that were fully tyrosinated or detyrosinated, and examined the suitability of these tracks for motility of recombinant GFP-tagged CENP-E motor. Using fluorescence microscopy we show that single molecules of CENP-E walk faster and more processively on detyrosinated microtubules. Moreover, on these tracks the CENP-E motor can generate larger force than on the tyrosinated microtubules, as determined using stationary optical trap. On both types of microtubules CENP-E took 8-nm steps, exhibited similar dwell times and frequencies of backward stepping. However, motor's detachment increased with resisting force faster when CENP-E was walking on tyrosinated microtubules, leading to the detachment from these polymers at on average smaller load, 4.5 pN vs. 6.4 pN for detyrosinated microtubules. The enhanced motility of CENP-E motor on detyrosinated microtubules, most notably its ability to carry a larger load, could potentially explain the targeted transport of mitotic chromosomes toward the spindle equator.
Didier Job - One of the best experts on this subject based on the ideXlab platform.
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Cap-Gly proteins at microtubule plus ends: is EB1 Detyrosination involved?
PLOS ONE, 2012Co-Authors: Anouk Bosson, Didier Job, Annie Andrieux, Jean-marc Soleilhac, Odile Valiron, Marie-jo MoutinAbstract:Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids −EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can Detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that Detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin.
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Motor-dependent microtubule disassembly driven by tubulin tyrosination
Journal of Cell Biology, 2009Co-Authors: Leticia Peris, Michael Wagenbach, Laurence Lafanechère, Jacques Brocard, Ayana T. Moore, Frank Kozielski, Didier Job, Linda Wordeman, Annie AndrieuxAbstract:In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of alpha-tubulin. The significance of this selective Detyrosination of MTs is not understood. In this study, we report that tubulin Detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)-inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the Detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.
Anouk Bosson - One of the best experts on this subject based on the ideXlab platform.
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vasohibins svbp are tubulin carboxypeptidases tcps that regulate neuron differentiation
Science, 2017Co-Authors: Leticia Peris, Anouk Bosson, Christophe Bosc, Pierre Heemeryck, Chrystelle AillaudAbstract:Reversible Detyrosination of α-tubulin is crucial to microtubule dynamics and functions, and defects have been implicated in cancer, brain disorganization, and cardiomyopathies. The identity of the tubulin tyrosine carboxypeptidase (TCP) responsible for Detyrosination has remained unclear. We used chemical proteomics with a potent irreversible inhibitor to show that the major brain TCP is a complex of vasohibin-1 (VASH1) with the small vasohibin binding protein (SVBP). VASH1 and its homolog VASH2, when complexed with SVBP, exhibited robust and specific Tyr/Phe carboxypeptidase activity on microtubules. Knockdown of vasohibins or SVBP and/or inhibitor addition in cultured neurons reduced detyrosinated α-tubulin levels and caused severe differentiation defects. Furthermore, knockdown of vasohibins disrupted neuronal migration in developing mouse neocortex. Thus, vasohibin/SVBP complexes represent long-sought TCP enzymes.
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a la recherche de l enzyme de Detyrosination du c terminus de l α tubuline
2013Co-Authors: Anouk BossonAbstract:Dans les cellules, les microtubules interviennent dans de nombreux evenements comme le maintien de l'architecture, la division du materiel genetique, la migration cellulaire ou encore le transport de vesicules et d'organites. Les modifications post-traductionnelles du le C-terminus de la tubuline, bloc de base des microtubules, apparaissent comme tres impliquees dans la regulation de ces fonctions car elles regulent le recrutement de leurs nombreux partenaires proteiques. Durant ma these, je me suis particulierement interessee a une de ces modifications post traductionnelles : le cycle de Detyrosination/tyrosination du C-terminus de l'α-tubuline. Ce cycle implique deux enzymes la Tubuline CarboxyPeptidase (TCP), qui clive la tyrosine a l'extremite de l'α-tubuline, et la Tubuline Tyrosine Ligase (TTL) qui re-additionne une tyrosine a la tubuline detyrosinee. Des etudes menees sur ce cycle et notamment la decouverte de la TTL ont permis de montrer que la presence d'une tyrosine a l'extremite C-terminale de l'α-tubuline est indispensable au developpement neuronal et que son absence favorise la progression tumorale. La TCP quant a elle est encore inconnue et sa decouverte apparait essentielle afin de pouvoir apprehender le cycle de Detyrosination/tyrosination dans sa globalite. Avec pour fil rouge l'identification de la TCP, mon travail s'est deroule en trois temps. Je me suis tout d'abord interessee a la proteine EB1. Cette proteine se lie a l'extremite positive des microtubules ou elle recrute de nombreux partenaires microtubulaires. EB1, presente le meme C-terminus que l'α-tubuline et notamment une tyrosine terminale indispensable a la liaison des proteines a CAP-Gly. Dans des cellules et des tissus sains j'ai montre qu'EB1 n'existe pas sous forme detyrosinee ce qui souligne la specificite de la TCP pour l'α-tubuline. Dans un second temps, j'ai participe a l'etude d'une famille de carboxypeptidases cytosoliques impliquees dans la neurodegenerescence, les CCPs, parmi lesquelles nous pensions trouver la TCP. Nous avons montre que quatre de ces enzymes (CCP1, 4, 5, et 6) retirent les glutamates presents de maniere laterale au C-terminus de la tubuline. Les CCP1, 4 et 6 peuvent egalement cliver le dernier glutamate de la tubuline detyrosine generant de l'α-tubuline ou les deux derniers acides amines ont ete clives (tubuline-Δ2). Aucune de ces carboxypeptidases ne se revelant etre la TCP, j'ai mis en place une methode biochimique dans le but de purifier cette enzyme. Apres plusieurs etapes de purification a partir de cerveaux de souris, des preparations enrichies en activite carboxypeptidase ont ete obtenues. Les analyses spectrometriques et bioinformatiques de ces preparations ont permis d'isoler des candidats TCP actuellement testes pour leur potentielle activite de Detyrosination du C-terminus de l'α-tubuline. Si la TCP n'est pas presente parmi eux, les outils developpes lors de cette etude devraient permettre une tres prochaine identification de cette enzyme essentielle.
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Cap-Gly proteins at microtubule plus ends: is EB1 Detyrosination involved?
PLOS ONE, 2012Co-Authors: Anouk Bosson, Didier Job, Annie Andrieux, Jean-marc Soleilhac, Odile Valiron, Marie-jo MoutinAbstract:Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids −EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can Detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that Detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin.
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Cap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination
2012Co-Authors: Anouk Bosson, Annie Andrieux, Jean-marc Soleilhac, Odile Valiron, Marie-jo MoutinAbstract:Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with a-tubulin and EB1 through their C-terminal amino acids 2EEY. Detyrosination (cleavage of the terminal tyrosine) of a-tubulin by tubulincarboxypeptidase abolishes CLIP170 binding. Can Detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of a-tubulin was only barely detyrosinated. Our results indicate that Detyrosination regulates CLIPs interaction with a-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin